GSM7712872: Drug treated cells from sample FB209 (LUAD pleural effusion), multiplexed RNA-seq, plate 1; Homo sapiens; RNA-Seq
Cells after drug perturbation were lysed in a 96-well plate using TriZol-LS. RNA was extracted using a Zymo Direct-zol-96 extraction kit. RNA-seq libraries were prepared using a customized version of the DRUG-seq protocol (Ye et al., 2018, PMID  PMC9207813) with the following modifications: 1) we changed the reverse transcription (RT) primer sequences to make them compatible with standard Illumina sequencing primers, 2) we start from purified RNA rather than a cell lysate, in our hands this resulted in less gDNA contamination of the sequencing libraries and simplified sample storage, 3) we optimized the protocol for ultra-low input samples by using a Zymo IC column to purify cDNA after RT and eluting in only 10 μl of hot (80C) water, and by using the Nextera XT kit (Illumina FC-131-1096) for library preparation; 4) we optimized the unique mapping rates of our libraries by performing a stringent size selection with one 0.5X cleanup to remove large fragments followed by two times 0.6X cleanup using KAPA pure beads (KAPA KK8000) resulting in fragment sizes between 500-800bp. RNA-seq  (3' digital gene expression)